Single‐Channel Recording of Glutamate Receptors

Chris Shelley1

1 Present address: Department of Biology, Franklin and Marshall College, Lancaster, 17604, Pennsylvania
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 11.16
DOI:  10.1002/0471141755.ph1116s68
Online Posting Date:  March, 2015
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Abstract

Highlighted in this unit are issues that should be considered when recording glutamate receptors at the single‐channel level, including some commonly encountered problems and their remedies. “UNIT 11.17, Single‐Channel Analysis of Glutamate Receptors” describes analysis techniques used to characterize the recorded single‐channel properties. © 2015 by John Wiley & Sons, Inc.

Keywords: single channel; glutamate receptor; ion channel

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Recording Single Glutamate Receptor Currents from Outside‐Out Patches
  • Support Protocol 1: Expression of Glutamate Receptor Channels in a Human Cell Line
  • Support Protocol 2: Fabrication of Recording Pipets
  • Reagents and Solutions
  • Commentary
  • References
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Recording Single Glutamate Receptor Currents from Outside‐Out Patches

  Materials
  • Glutamate or other appropriate agonist (e.g., Sigma‐Aldrich, Tocris Bioscience)
  • Extracellular solution (see recipe)
  • Intracellular solution (see recipe)
  • Mammalian cells expressing glutamate receptors, on coverslips in wells of 4‐well plates ( protocol 2)
  • Small (e.g., 1‐ml) syringe
  • 0.22‐μm syringe filter (e.g., EMD Millipore)
  • Microfil pipet filler (World Precision Instruments)
  • Electrophysiology rig including:
    • Air table (e.g., Automate Scientific, Thorlabs, TMC)
    • Faraday cage (e.g., Automate Scientific, Harvard Apparatus, Thorlabs)Recording chamber and stage (e.g., Scientifica)
    • Pipet holder (e.g., G23 Instruments, Molecular Devices)
    • Coarse micromanipulator (e.g., Narashige, Warner Instruments, World Precision Instruments).
    • Fine micromanipulator (e.g., Narashige, Warner Instruments, World Precision Instruments)
    • Patch clamp amplifier and headstage (e.g., Heka Elektronik, Molecular Devices, Warner Instruments)
    • Digitizer (e.g., Cambridge Electronic Design, Molecular Devices)
    • Fluorescent microscope (e.g., Leica Microsystems, Nikon Instruments, Olympus, Zeiss)
  • Recording pipets ( protocol 3)
  • Computer for data storage and analysis
  • Data acquisition and analysis software (see Table 11.16.1 in unit 11.17)

Support Protocol 1: Expression of Glutamate Receptor Channels in a Human Cell Line

  Materials
  • Lipofectamine transfection reagent (Life Technologies)
  • Opti‐MEM transfection medium (Life Technologies)
  • cDNA encoding glutamate receptor ion channel subunits (e.g., Origene)
  • cDNA encoding green fluorescent protein (e.g., Origene)
  • HEK 293 cells (e.g., American Type Tissue Collection, European Collection of Cell Cultures)
  • Ca2+‐ and Mg2+‐free phosphate‐buffered saline (Dulbecco's PBS; e.g., Life Technologies)
  • Trypsin/EDTA solution (0.5 g porcine trypsin, 0.2 g tetrasodium EDTA per 1000 ml; e.g., Life Technologies; can be stored for many months in 1‐ml aliquots at −20°C)
  • Dulbecco's modified Eagle medium (DMEM) with glutamine and low glucose (e.g., Life Technologies, Sigma‐Aldrich)
  • HEK cell culture medium (see recipe)
  • 25‐cm2 tissue culture flasks, (e.g., Life Technologies)
  • 15‐ml conical centrifuge tubes (e.g., Life Technologies)
  • Clinical benchtop centrifuge (e.g., Fisher Scientific)
  • 10‐mm poly‐L‐lysine‐coated coverslips (see recipe)
  • Four‐well plates (e.g., Life Technologies, VWR)

Support Protocol 2: Fabrication of Recording Pipets

  Materials
  • Sylgard 184 silicone elastomer (Dow Corning)
  • Standard wall (1.5 mm outer diameter, 0.86 mm inner diameter) borosilicate glass capillary tubes, 150 mm in length (Warner Instruments)
  • Pipet puller (e.g., Narashige, Sutter Instrument Company, Warner Instruments)
  • Dissecting microscope for application of elastomer
  • Heat gun or heated coil for curing the elastomer
  • Microforge (e.g., Heka Elektronik, Narashige, Warner Instruments)
  • NOTE: Please refer to other units in Current Protocols in Pharmacology Chapters 10 and 11 for additional detail on pipet pulling.
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Figures

Videos

Literature Cited

References
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